This invention relates to a spectrofluorometer, and more particularly to a spectrofluorometer which can measure an emission polarization spectrum precisely and simply.
The polarization measurement which has been carried out in a prior-art spectrofluorometer affords an excitation polarization spectrum. More specifically, an excitation spectrum signal I.sub.VV (.lambda..sub.EX) is obtained by performing the wavelength scanning of an excitation monochromator under the conditions under which an emission wavelength is fixed and under which the polarization direction of an excitation polarizer is set to be vertical, while the polarization direction of an emission polarizer is set to be vertical. An excitation spectrum signal I.sub.VH (.lambda..sub.EX) is obtained by performing the wavelength scanning of the excitation monochromator with the polarization direction of the excitation polarizer set vertical and the polarization direction of the emission polarizer set horizontal. From these excitation spectrum signals, the excitation polarization spectrum P (.lambda..sub.EX) is found in conformity with the following expression: ##EQU2## where G denotes the grating correction factor for correcting the polarization characteristics of an emission monochromator dependent upon the measurement wavelength. The factor G is obtained from the following expression: EQU G=I.sub.HV /I.sub.HH
The evaluation of the factor G is executed by operations described hereunder, by exploiting the fact that when excitation light is polarized in the horizontal direction, fluorescence observed on a horizontal plane in a direction of 90 degrees relative to the excitation light is not polarized.
The emission or fluorescence intensity signal I.sub.HH is measured at the time when, at the emission wavelength (fixed) for use in the measurement, the polarization direction of the excitation polarizer is made horizontal and that of the emission polarizer is made horizontal. Subsequently, the emission intensity signal I.sub.HV is measured at the time when, at the same emission wavelength, the polarization direction of the excitation polarizer is made horizontal and that of the emission polarizer is made vertical. The grating correction factor G for correcting the polarization characteristics of the emission monochromator at the emission wavelength used in the measurement is obtained from these signals I.sub.HH and I.sub.HV.
As described above, in the prior-art spectrofluorometer, the excitation polarization spectrum is measured under the condition that the emission wavelength is constant, in other words, that the polarization characteristics of the emission monochromator are constant. A change in the emission wavelength results in changing the grating correction factor G accordingly.
On the other hand, the advent of a spectrofluorometer which can measure not only the excitation polarization spectrum but also an emission polarization spectrum has been wished for owing to the desire of investigating properties inherent to a substance from various angles. The reason is that samples on which the properties inherent to the substance cannot be qualitatively grasped merely by the measurement of the excitation polarization spectrum have been often met with.
The measurement of the emission polarization spectrum is carried out by fixing an excitation wavelength instead of fixing the emission wavelength in the measurement of the excitation polarization spectrum and by scanning the emission wavelength instead of scanning the excitation wavelength. Accordingly, a problem in the case of measuring the emission polarization spectrum with the prior-art apparatus is that the scanning of the emission wavelength causes the grating correction factor G for correcting the polarization characteristics of the emission monochromator dependent upon the measurement wavelength, to vary depending upon the scanning wavelength. It requires much labor and a long time to evaluate the grating correction factor G at various wavelength positions of the scanning wavelength. Therefore, the emission polarization spectrum has been obtained without considering the grating correction factor G, or the measurement of the emission polarization spectrum has been given up. The emission polarization spectrum obtained without taking the grating correction factor G into account cannot be said the true emission polarization spectrum, and its precision being too low has made it impossible to be supplied to the qualitative analysis.